模拟昆虫中肠环境对球孢白僵菌分生孢子的影响

Impact of insect midgut-like environment on conidia of entomopathogenic fungus Beauveria bassiana

为了观察孢子在模拟昆虫中肠环境的变化,先配制不同组分的中肠模拟消化酶液(均为0.5%),将稳定表达绿色荧光蛋白菌株的分生孢子悬浮其中,25℃或35℃恒温孵育4h后,分别采用流式细胞仪和扫描电子显微镜检测孢子荧光强度和孢子壁形态的变化.孢子荧光强度的改变可以反映酶液对孢子壁消解的程度.结果显示,5种模拟消化液导致荧光强度显著上升,分别为蛋白酶K、链霉蛋白酶E、纤维素酶、胰蛋白酶和α-淀粉酶,但昆布多糖酶、葡萄糖苷酶、蜗牛酶模拟消化液导致荧光强度下降.与单因素模拟消化液相比,复合酶类模拟消化液作用后的荧光强度大幅下降.在pH5～11范围内,复合酶类模拟消化液处理后的分生孢子荧光强度变化情况差异显著.扫描电子显微镜观察到分生孢子经昆虫模拟消化液处理后孢壁表面结构发生明显变化.这意味着昆虫中肠消化液可能影响球孢白僵菌分生孢子壁结构及其侵染功能.

The conidia of a transgenic strain （Bb2860G6） that steadily expresses green fluorescence protein were suspended in the solutions of 0.5% enzymes similar to those present in different insect midguts and then incubated at 25℃ or 35℃ for 4 h, followed by examining the fluorescence intensity of conidia （i. e. , cell permeability） through flow cytometry and the structural changes of conidial wall under scanning electron microscope. The structural changes of conidial wall treated by different enzymes were associated with variation of the conidial fluorescence intensity. As a result, the fluorescence intensity of the treated conidia was significantly enhanced by treatments with proteinase K, pronase E, cellulase, trypsin and a-amylase respectively, but reduced by laminarinase, glucosidase and snailase. A mixture of 0.5 % proteinase K with 0.5 % cellulase or 0.5 % trypsin with 0.5 % cellulase decreased the fluorescence intensity more than a single enzyme, and the decreases varied significantly at pH 5-11, which occur in the midgut environments of different insects. Conspicuous changes of the conidial wall were also observed after 4 h treatment with the digestive enzymes. The results imply that insect midgut environment may affect the wall structure and activity of B. bassiana conidia.